SUMMARY High-grade serous ovarian cancer (HGSOC) frequently harbors defects in DNA repair pathways that confer homologous recombination (HR) repair deficiency, as well as compromised stability of stalled DNA replication forks. Such defects frequently involve BRCA alterations and confer sensitivity to inhibitors of poly (ADP-ribose) polymerase (PARP) inhibitors. These agents have now entered the routine HGSOC armamentarium both in the advanced and maintenance settings. An overarching objective of this project is to address the emerging problem of PARP inhibitor resistance that will assume greater importance as PARP inhibitor use increases. The project focuses on inhibition of Ataxia telangiectasia and Rad3-related (ATR) as a strategy designed to reverse the two major mechanisms of acquired PARP inhibitor resistance, including restoration of HR repair and stabilization of DNA replication forks. Three Specific Aims are proposed. In Aim 1, we will assess the activity and mechanisms of the ATR inhibitor AZD6738 as monotherapy and in combination the PARP inhibitor olaparib in in vitro and in vivo BRCA-mutated cell line, organoid culture and patient-derived xenograft (PDX) models of HGSOC with acquired PARP inhibitor resistance. This work will prepare the way for a clinical trial in Aim 2 combining AZD6738 and olaparib with a schedule that maximizes ATR inhibition in patients with PARP inhibitor-resistant BRCA- mutated HGSOC. Once the maximum tolerated doses are established, we will confirm tolerability and assess preliminary antitumor activity in an expansion cohort of twelve patients. Paired biopsies will be procured for proof-of-mechanism pharmacodynamic studies in which HR is assessed with an immunohistochemical RAD51 assay and replication fork stability is assessed with a DNA fiber assay in organoid cultures. In Aim 3, we will determine the activity of ATR inhibition alone and in combination with gemcitabine in HGSOCs with a high degree of replication stress but are intrinsically unresponsive to PARP inhibition. AZD6738 and gemcitabine will be studied in cell line, organoid and PDX models harboring high-level CCNE1 or MYC amplification. We will analyze replication stress in these models and its exacerbation by ATR inhibition and gemcitabine using immunohistochemical, cytological and gene expression signature biomarkers of ATR pathway activation. Lastly, we will leverage a recently completed study of gemcitabine vs. gemcitabine combined with the ATR inhibitor M6620 in platinum-resistant HGSOC patients, in which the combination was superior among patients with a platinum-free interval of less than 3 months. We will test the hypothesis that this group was enriched with tumors carrying a high degree of replicative stress, defined by CCNE1 or MYC amplification, as well as by biomarkers developed in the preclinical models. Taken together, these aims will allow us to establish a role for ATR inhibition in the HGSOC armamentarium, identify new therapeutic avenues for HGSOC populations with poor prognosis and provide insights into the biology of PARP inhibitor resistance.